Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g., cholera toxin, pertussis toxin). Similar to cholera toxin, some mammalian ADP-ribosyltransferases specifically use the guanidino group of arginine as an ADP-ribose acceptor. Five mammalian NAD: arginine ADP-ribosyltransferases (ART) were cloned from various tissues. The ART1 proteins were shown to be cell surface proteins, linked through a glycosylphosphatidylinositol (GPI) anchor. The transferases appear to be selectively expressed in mammalian tissues. ART-1 is found in skeletal and cardiac muscle and lymphoid cells, ART-2 in lymphocytes, ART-4 in spleen and ART-5 in testis. In view of the expression of ARTs in tissues involved in immunoregulation, we examined whether ARTs might be expressed in pulmonary epithelial cells. Human airway epithelial cells were isolated by bronchoalveolar lavage and bronchial brushing. By in situ analysis and Northern blot, ART1 mRNA was identified in airway epithelial cells. As expected for GPI-anchored proteins, the localization of ART1 at the apical surface of ciliated epithelial cells was demonstrated by staining with polyclonal anti-ART1 antibody and confirmed by loss of this immunoreactivity after treatment with phosphatidylinositol-specific phospholipase C(PI-PLC), which selectively cleaves GPI-anchors and releases proteins from the plasma membrane. By in situ hybridization with specific ART3 and ART4 oligonucleotides, two additional members of the RT6 superfamily were also demonstrated in epithelial cells. In agreement with these findings, ART3 and ART4 mRNAs were identified by RT-PCR of poly(A)+RNA from human trachea. Interestingly, these proteins appeared to be preferentially localized to the airway epithelium. The localized expression of these members of the RT6 superfamily in human pulmonary epithelial cells may reflect a role in cell-cell signaling during immune responses within the airway.